The goal of this project is to develop, through repertoire cloning technology, a panel of broadly neutralizing human anti-HIV-1 envelope glycoprotein antibodies (Abs), to characterize their affinity and epitope specificity and to perform a detailed molecular analysis of antibody gene structure. We will first characterize the Ab response in HIV-1 infected patients. Total serum anti-gp120 and anti-gp41 Abs will be purified by affinity chromatography on gp160- and then on gp120-Sepharose and the latter eluate then further purified to obtain CD4-site, non-CD4 site, V3 loop and non V3 loop specific Ab fractions. These different fractions will be tested for both type-restricted and broadly reactive neutralizing activities using both primary isolates and several laboratory strains of HIV-1. We will use the human monoclonal antibody (MAb) F105 which blocks gp120/CD4 binding and which we have cloned and expressed in bacteria to further characterize the CD4-site specific Abs for their ability to block F105 binding to the F105 epitope. In selected patients with these broadly neutralizing serum Abs, we will use recombinant DNA techniques to produce hierarchial (F105 idiotype) and random combinatorial libraries of immunoglobulin genes from their peripheral blood lymphocytes, bone marrow or surgical specimens. We will express these rearranged immunoglobulin genes as single chain Fv antibody (sFv)-gene III fusion proteins (phage antibodies). This powerful technology will enable us to affinity select those phage which express sFv reactive with HIV-1 gp120/gp41. These phage will be amplified, individual clones isolated and the antibody genes will be DNA sequenced. We will further characterize these gp120/gp41 reactive phage Abs for gp120/gp41 binding affinity, epitope reactivity as well as for HIV neutralization and syncytia inhibition. We will perform a detailed genetic analysis of the VDJ (for heavy chain) and VJ (for light chain) gene rearrangement and will correlate Ab molecular structure with gp120/gp41 epitope reactivity, determine germ line gene usage and identify somatic mutations which may have arisen. Lastly, we will engineer these phage Abs to be expressed in native form with human IgG and IgM expression vectors transfected into myeloma cells. Achieving these objectives and goals will lead to a more complete understanding of the specific role of broadly neutralizing anti- HIV-1 envelope glycoprotein Abs in modulating HIV infection and of the molecular nature of this Ab response. In addition, these studies may lay the foundation for passive immunoprophylaxis following exposure, in preventing the vertical transmission of HIV and for immunotherapy in HIV- infected patients using engineered anti-HIV-1 envelope glycoprotein MAbs.